Phytochemistry and biosynthesis of δ-lactone withanolides

Withanolides are highly oxygenated natural products. These C₂₈ steroids with ergostane-based skeletons functionalized at C-22 and C-26 form six-membered δ-lactone rings. Withanolides containing a δ-lactone side chain often occur in Solanaceae and have a variety of biological activities because of their complicated structures. Characteristic spectroscopic behaviors and biosynthesis of withanolides are conducive to their structural elucidation and “biomimetic synthesis”, respectively. However, the last review to summarize their spectroscopic features and biosynthesis was in 1996. Since then, many withanolides with novel structures have been described by their spectra with biosynthesis investigated with many bioassays. This review surveys δ-lactone withanolides and emphasizes their spectral features, configurations and biosynthetic genes. The period reviewed includes through January 2014. We also include phytochemical species.     

The application of δ¹⁸O and δD for understanding water pools and fluxes in a Typha marsh

The δ¹⁸O and δD composition of water pools (leaf, root, standing water and soil water) and fluxes [transpiration (T), evaporation (E)] were used to understand ecohydrological processes in a managed Typha latifolia L. freshwater marsh. We observed isotopic steady‐state T and deep rooting in Typha. The isotopic mass balance of marsh standing water showed that E accounted for 3% of the total water loss, T accounted for 17% and subsurface drainage (D) accounted for the majority (80%). There was a vertical gradient in water vapour content and isotopic composition within and above the canopy sufficient for constructing an isotopic mass balance of water vapour during some sampling periods. During these periods, the proportion of T in evapotranspiration (T/ET) was between 56 ± 17% and 96 ± 67%, and the estimated error was relatively high (>37%) because of non‐local, background sources in vapour. Independent estimates of T/ET using eddy covariance measurements yielded similar mean values during the Typha growing season. The various T/ET estimates agreed that T was the dominant source of marsh vapour loss in the growing season. The isotopic mass balance of water vapour yielded reasonable results, but the mass balance of standing water provided more definitive estimates of water losses.     

The isolation and properties of δ-tocotrienol from Hevea latex

1. delta-Tocotrienol (8-methyltocotrienol) was isolated from the latex of Hevea brasiliensis. This new member of the tocopherol family is a pale-yellow oil at room temperature. 2. The properties of delta-tocotrienol are very similar to those of delta-tocopherol and the small differences can be explained by the change in side chain. 3. The ultraviolet and infrared spectra of delta-tocotrienol were determined and a conversion factor for use with the Emmerie-Engel reaction was worked out. Details are given for the chromatography of delta-tocotrienol on thin layers (adsorption and partition) and reversed-phase paper, and the nitroso derivatives were formed. 4. An ethyl carbonate ester of delta-tocotrienol was prepared and compared with a similar ester of delta-tocopherol. 5. Hydroxymethylation of delta-tocotrienol followed by reduction gave beta-tocotrienol as a major product.     

Conservation of δ-crystallin gene structure between ducks and chickens

A cloned chicken delta-crystallin cDNA was used to identify two putative delta-crystallin genes in the duck by Southern blot hybridization. A DNA fragment containing most of one of these genes was isolated from a library made in bacteriophage lambda Charon 28A containing genomic DNA from 14-day-old embryonic ducks. Electron microscopy, partial gene sequencing, primer extension analysis using duck mRNA, and comparison with the well-characterized chicken delta-crystallin genes suggest that our cloned duck delta-crystallin gene, like the chicken delta-crystallin genes, is 8-10 kb long and contains 17 exons. Hybridization and sequencing data show great similarity between the homologous 5' untranslated and coding exons of the duck and chicken delta-crystallin genes. Overall, the homologous introns also appear to have approximately 30% sequence similarity, and have been subject to deletion/insertion events. Our partial characterization of duck delta-crystallin gene sequences suggests that this avian and reptilian crystallin family has been conserved during evolution, as have the other crystallin gene families that are expressed in the eye lens.     

Reliability of δ13C and δ15N in faeces for reconstructing savanna herbivore diet

We tested the reliability of herbivore faecal δ¹³C and δ¹⁵N values for reconstructing diet through review of an extensive database derived from a 3-year study of ungulates in South Africa's Kruger National Park. Faeces are a useful material for stable isotope studies of diet because they record dietary turnover at very short time scales, and because sampling is non-invasive. However, the validity of faecal isotope proxies may be questioned because they represent only undigested food remains. Results from Kruger Park confirm that free-ranging browsers have faecal δ¹³C consistent with C₃ feeding, grazer faeces are C₄, and mixed-feeder faeces intermediate. Although the respective ranges do not overlap, there is significant variation in faecal δ¹³C of browsers and grazers (～2.0–4.0‰) across space and through time. We demonstrate that most (～70%) of this variation can be ascribed to corresponding patterns of variation in the δ¹³C of C₃ and C₄ plants, respectively, re-enforcing the fidelity of faecal isotope proxies for diet but highlighting a need for mixing models that control for variations in plant δ¹³C in order to achieve accurate diet reconstructions. Predictions for the effects of climate (rainfall) and ecophysiology on ¹⁵N-abundance variations in mammals do not persist in faeces. Rather, faecal δ¹⁵N tracks changes in plant δ¹⁵N, with further fractionation occurring primarily due to variations in dietary protein (reflected by %N). Controlling for these effects, we show that a dual-isotope multiple source mixing model (Isosource) can extend diet reconstructions for African savanna herbivores beyond simplified C₃/C₄ distinctions, although further understanding of variations in mammal δ¹⁵N are needed for greater confidence in this approach.     

Differences in the C age, δC and δO of Holocene tufa and speleothem in the Dinaric Karst

We studied Holocene speleothems and tufa samples collected in numerous caves and rivers in the Dinaric Karst of Croatia, Slovenia, Bosnia and Herzegovina, as well as Serbia and Montenegro. Differences in the formation process of tufa and speleothems are discussed in the context of their isotopic composition (¹⁴C, ¹³C and ¹⁸O), as well as the chemistry of surface water (rivers, lakes) and drip water (in caves). The physical and chemical parameters monitored in the surface water (tufa precipitation) and drip water (speleothem precipitation) show that more stable conditions accompany speleothem rather than tufa formation. This is particularly obvious in the water temperature variations (2-22°C in surface water and 7-12°C in drip water) and in saturation index variation (3-11 in surface water and 1-6 in drip water). The range of ¹⁴C ages recorded by Holocene speleothems (∼12?000 yr) is wider by several thousands years than that of Holocene tufa samples (∼6000 yr). δ¹³C values for tufa samples range from −12‰ to −6‰ and for speleothem samples from −12‰ to +3‰ reflecting higher soil carbon and/or vegetation impact on the process of tufa than on speleothem formation. The differences in δ¹⁸O values of tufa and speleothem samples from different areas reflect different temperature conditions and differing isotopic composition in the water. The study shows that speleothems from the Dinaric Karst can be used as global palaeoclimatic records, whereas tufa records changes in the local palaeoenvironment.     

Enantioselective Dehydrochlorination of δ-Hexachlorocyclohexane and δ-Pentachlorocyclohexene by LinA1 and LinA2 from Sphingobium indicum B90A

δ-Hexachlorocyclohexane (δ-HCH), one of the prevalent isomers of technical HCH, was enantioselectively dehydrochlorinated by the dehydrochlorinases LinA1 and LinA2 from Sphingobium indicum B90A to the very same δ-pentachlorocyclohexene enantiomer. Racemic δ-pentachlorocyclohexene, however, was transformed with opposite enantioselectivities by the two enzymes. A transformation pathway based on an anti-1,2-elimination, followed by a syn-1,4-elimination and a subsequent syn-1,2-elimination is postulated.     

Influence of Bt-plants on soil biota and pleiotropic effect of δ-endotoxin-encoding genes

The critical review of experimental conclusions stating environmental safety of transgenic plants reveals that the methods and test materials employed in some studies were inadequate to the goals pursued. A large-scale application of transgenic Bt-plants may result in long-lasting negative impact on the environment. First, the cultivation of these plants leads to accumulation of Bt-toxins in soil. Second, the decomposition of transgenic plants takes significantly longer time compared to that of isogenic lines. Third, the biological activity of soils under transgenic crops is lower than in the control plots. The transfer of δ-endotoxin-encoding genes to the genome of agricultural crops affects simultaneously several entirely different traits of genetically modified plants, thus exerting pleiotropic effects. This gives rise to a paradoxical situation: the genetically engineered crops selected on the trait of resistance to herbivorous insects of the order Lepidoptera become more attractive for herbivores from the other order, Homoptera.     

Temporal records of δ13C and δ15N in North Pacific pinnipeds: inferences regarding environmental change and diet

Sea lion and seal populations in Alaskan waters underwent various degrees of decline during the latter half of the twentieth century and the cause(s) for the declines remain uncertain. The stable carbon (¹³C/¹²C) and nitrogen (¹⁵N/¹⁴N) isotope ratios in bone collagen from wild Steller sea lions (Eumetopias jubatus), northern fur seals (Callorhinus ursinus) and harbor seals (Phoca vitulina) from the Bering Sea and Gulf of Alaska were measured for the period 1951-1997 to test the hypothesis that a change in trophic level may have occurred during this interval and contributed to the population declines. A significant change in '¹⁵N in pinniped tissues over time would imply a marked change in trophic level. No significant change in bone collagen '¹⁵N was found for any of the three species during the past 47 years in either the Bering Sea or the Gulf of Alaska. However, the ¹⁵N in the Steller sea lion collagen was significantly higher than both northern fur seals and harbor seals. A significant decline in '¹³C (almost 2 ‰ over the 47 years) was evident in Steller sea lions, while a declining trend, though not significant, was evident in harbor seals and northern fur seals. Changes in foraging location, in combination with a trophic shift, may offer one possible explanation. Nevertheless, a decrease in '¹³C over time with no accompanying change in '¹⁵N suggests an environmental change affecting the base of the foodweb rather than a trophic level change due to prey switching. A decline in the seasonal primary production in the region, possibly resulting from decreased phytoplankton growth rates, would exhibit itself as a decline in '¹³C. Declining production could be an indication of a reduced carrying capacity in the North Pacific Ocean. Sufficient quantities of optimal prey species may have fallen below threshold sustaining densities for these pinnipeds, particularly for yearlings and subadults who have not yet developed adequate foraging skills.     

Research article: small-scale spatial variation of δ13C and δ15N isotopes in Antarctic carbon sources and consumers

Regional food web studies that fail to account for small-scale isotopic variability can lead to a mismatch between an organism’s inferred and true trophic position. Misinterpretation of trophic status may result, substantially limiting spatial and temporal comparability of food web studies. We sampled several carbon sources and consumers in a nested design to assess the variability of food web members across small spatial scales (100 s of m to several km) in regions around the Windmill Islands and Vestfold Hills in East Antarctica. For carbon sources, δ¹³C in sea ice POM was particularly variable between locations (km apart) and between sites (100 s of m apart) with replicate samples varying by up to 16‰. Macroalgae δ¹³C was less variable (replicate samples ranging up to 6.9‰ for the red alga Iridaea cordata), yet still differed between locations. Sediment POM and pelagic POM were the least variable, displaying minimal differences between locations or sites for δ¹³C and δ¹⁵N. Three out of eight consumers were significantly different between locations for δ¹³C, and five out of eight for δ¹⁵N, with the fish Trematomus bernacchii the most variable for both δ¹³C and δ¹⁵N. At smaller scales, the amphipod Paramorea walkeri showed significant variation between sites in δ¹³C but not in δ¹⁵N. We attribute small-scale variability to the dynamic physical environment for carbon sources in coastal systems and a close coupling of diet to habitat for consumers. We highlight the need to account for small-scale spatial variation in sampling designs for regional food web studies.     

Binding of δ9-tetrahydrocannabinol and diazepam to human serum albumin

Cannabis is the most commonly used illicit drug worldwide. Cannabis users also appear to use other psychoactive drugs more frequently than noncannabis users. Here, Δ9-tetrahydrocannabinol (THC) and diazepam binding to human serum albumin (HSA) and HSA-heme is reported. THC binds to two different binding sites of HSA (K(d1) ≤ 10(-7) M and K(d2) = 10(-3)M) without affecting diazepam binding (K(d) = 1.2 × 10(-5) M). THC binding to the high-affinity site accounts for the low free fraction of the drug in plasma. Moreover, THC increases the affinity of heme for HSA. Accordingly, the affinity of THC for HSA-heme is higher than that for HSA. THC could bind to FA2 and FA7 sites, as substantiated by docking simulations; nevertheless, the observed allosteric effect(s) suggests that the primary binding site of THC is the FA2 cleft that positively modulates heme affinity. Possibly, the HSA conformational transition(s) induced by THC binding could account for drug delivery to the liver through receptor- mediated endocytosis.     

Purification and general properties of δ-aminolaevulate dehydratase from cow liver

1. delta-Aminolaevulate dehydratase, the enzyme catalysing the condensation of delta-aminolaevulic acid to porphobilinogen, has been prepared from cow liver and its properties have been studied. The enzyme has been purified 310-fold. 2. The purified preparation behaves as a single protein under gel filtration on Sephadex and Bio-Gel columns; it migrates as a single band in disk and starch-gel electro-phoresis at different pH values and it sediments as a single symmetrical peak in the ultracentrifuge. 3. The pH optimum for the pure enzyme was 6.8, the K(m) at pH 6.8 and 38 degrees was 1.5x10(-4)m, the isoelectric point was about pH 4.9 and the molecular weight was 140000+/-14000 by the gel-filtration method. Maximal enzyme activity was observed at 65 degrees . 4. The presence of thiol groups in the enzyme system, essential for its activity, was indicated and the total number of thiol groups was determined. 5. After the first steps of purification the enzyme required cysteine or reduced glutathione for activity.     

Effect of pyridoxal phosphate and cysteine on δ-aminolaevulinate synthetase and dehydratase in soya

The specific activity of ALA-S extracts prepared from lyophilized soya callus growing in light or dark showed a striking increase when tissue cultures were grown in the presence of pyridoxal phosphate (PyP) in order to complex aminothiols. By contrast ALA-D specific activity in the light grown callus was reduced on incorporation of PyP into the growth medium. Cysteine (Cy) added to the culture medium did not influence the specific activities of either enzyme.     

Kinetic and Sequence-Structure-Function Analysis of LinB Enzyme Variants with β- and δ-Hexachlorocyclohexane

Organochlorine insecticide hexachlorocyclohexane (HCH) has recently been classified as a ‘Persistent Organic pollutant’ by the Stockholm Convention. The LinB haloalkane dehalogenase is a key upstream enzyme in the recently evolved Lin pathway for the catabolism of HCH in bacteria. Here we report a sequence-structure-function analysis of ten naturally occurring and thirteen synthetic mutants of LinB. One of the synthetic mutants was found to have ∼80 fold more activity for β- and δ-hexachlorocyclohexane. Based on detailed biophysical calculations, molecular dynamics and ensemble docking calculations, we propose that the latter variant is more active because of alterations to the shape of its active site and increased conformational plasticity.     

Synthesis and biological evaluation of optically active conjugated γ- and δ-lactone derivatives

An efficient synthesis of racemic and both enantiomeric forms of heteroaryl substituted γ- and δ-lactone derivatives derived from allyl and homoallyl alcohol backbones has been accomplished via ring closing metathesis reaction. 2-Heteroaryl substituted allyl and homoallyl alcohols have been efficiently resolved through enzymatic method with high ee (97-99%) and known stereochemistry. Antimicrobial and antioxidant activities of target lactones were evaluated.     

Lytic effects of melittin and δ-haemolysin from Staphylococcus aureus on vesicles of dipalmitoylphosphatidylcholine

The effects of the lytic peptides, melittin and δ-haemolysin, are compared in vesicles of gel-phase dipalmitoylphosphatidylcholine (DPPC), using calcein as trapped marker. At low concentration, both toxins cause vesicles to lose contents in 5 mM phosphate buffer near neutral pH, with melittin being the more active. As phosphate concentration is increased, the kinetics of melittin-induced leakage change from a slow, sustained loss to a rapid ‘burst’ of leakage when melittin is present mainly as tetramer in solution, under conditions where it is reported to lose haemolytic activity towards erythrocytes. At low phosphate concentration, the leakage induced by δ-haemolysin is preceded by a lag phase, though fluorescence measurements show that binding of toxin is rapid. At higher phosphate concentration, the toxin binds rapidly to vesicles, but causes no leakage of entrapped calcein. Steady-state fluorescence spectra show no obvious differences in tryptophan emission for δ-haemolysin bound to lipid in high- or low-phosphate buffer. Spin-label fluorescence-quenching studies show that the single tryptophan residue of δ-haemolysin is buried within the lipid bilayer at all phosphate concentrations used. In gel-phase DPPC, δ-haemolysin shows no tendency to cause vesicle aggregation over several hours, as judged by light scattering, though a slow non-linear effect is seen above the lipid phase transition temperature. These effects are contrasted with those of melittin under similar conditions.     

Generation of active oxygen species during coupled autoxidation of oxyhemoglobin and δ-aminolevulinic acid

The conversion of oxyhemoglobin to mathemoglobin has been shown via spectrophotometric, circular dichroism and polarographic studies to be accelerated by δ-aminolevulinic acid, a major heme-precursor accumulated in a number of heme-linked pathologies. Concomitantly, δ-aminolevulinic acid undergoes aerobic oxidation. The intermediacy of oxygen radicals in these processes was evidenced by the inhibitory effect of catalase, superoxide dismutase and mannitol. These results are relevant to the exacerbated production of active oxygen species in intermittent acute porphyria and saturnism carriers.     

Differences in δ15N and δ13C between embryonic and maternal tissues of the ovoviviparous bluntnose sixgill shark Hexanchus griseus

Stable nitrogen (δ¹⁵N) and carbon (δ¹³C) isotope ratios from muscle, liver and yolk were analysed from the mother and embryos of an ovoviviparous shark, Hexanchus griseus. Embryonic liver and muscle had similar δ¹⁵N and δ¹³C ratios or were depleted in heavy isotopes, compared to the same maternal somatic and reproductive yolk tissues, but no relationship existed between δ¹⁵N or δ¹³C and embryo length, as expected, because a switch to placental nourishment is lacking in this species. This study expands the understanding of maternal nourishment and embryonic stable isotope differences in ovoviviparous sharks.     

Ferrochelatase and δ-aminolaevulate synthetase in brain, heart, kidney and liver of normal and porphyric rats. The induction of δ-aminolaevulate synthetase in kidney cytosol and mitochondria by allylisopropylacetamide

1. delta-Aminolaevulate synthetase was detected in liver and kidney mitochondria prepared from normal rats. 2. The administration of allylisopropylacetamide induced an increase in delta-aminolaevulate synthetase in both liver and kidney mitochondria and the enzyme also appeared in the cytosol fraction of both tissues. Comparison with the distribution of glutamate dehydrogenase indicated that this soluble kidney delta-aminolaevulate synthetase was truly of cytosol origin and did not arise from disrupted mitochondria. The kidney cytosol enzyme was inhibited by 50% by 50mum-protohaem. 3. delta-Aminolaevulate synthetase could not be detected in mitochondria or cytosol from heart or brain from normal or porphyric rats. 4. The administration of allylisopropylacetamide caused little or no increase in ferrochelatase or cytochrome content of liver, kidney, heart or brain mitochondria.     

The environmental water of the middle Eocene Arctic: Evidence from δD, δ18O and δ13C within specific compounds

The extensive vertical exposure (> 150 m) of terrestrial sediments on Axel Heiberg Island, which contain thick fossiliferous lignites, presents an exceptional opportunity to follow the establishment and re-establishment of Arctic Metasequoia forests during the middle Eocene. We compared δD values in n-alkanes of chain length 23, 25, 27 and 29 with δ¹⁸O values in phenylglucosazone (P-G) derived from α-cellulose; we also analyzed %-abundance of ferns, gymnosperms and angiosperms using pollen and spores isolated from each lignite. Our results showed that forest composition was altered upon uplift, as gymnosperms became more abundant within the relatively well-drained upland sediments. This was also reflected in the small (1‰), but significant, increase in the δ¹³C value of TOM from lowland to upland environments. However, neither the δD values of n-alkanes nor the δ¹⁸O in P-G were statistically different in the upland sediments, as compared to the lowland sediments; from this we inferred that the oxygen isotope signature of environmental water available to the forests for plant growth was relatively uniform throughout the time of the fossil forests. The δD value of environmental water implied by both n-alkanes and P-G ranged from ? 168 to ? 131% and was considerably enriched compared to all environmental water samples available from the modern Arctic region (< ? 180%). In addition to indicating a warmer Eocene Arctic, subject to meteoric transport patterns different from today's, these results argue against the presence of an Eocene polar ice cap.